Our long-term objective is to gain a comprehensive understanding of the molecular mechanism by which gamma-aminobutyric acid (GABA) transporters accomplish Na+/Cl-/GABA cotransport across the plasma membrane. The GABA transporters use the Na + electrochemical gradient to transport GABA into cells after its release from nerve terminals and, thus, they regulate the concentration and lifetime of GABA in synaptic and extra-synaptic regions in the nervous system. In addition, they prevent spillover of GABA to surrounding synapses and, therefore, they ensure synaptic specificity. GABA is the most abundant inhibitory neurotransmitter in the central nervous system and, therefore, potentiation of GABAergic neurotransmission via inhibition or reversal of the GABA transporters is believed to have therapeutic value in treating epileptic seizures and stroke. Four GABA transporter isoforms are present in the mammalian brain (GAT1, GAT2, GAT3, and GAT4), and exhibit significant differences in function, pharmacology, and localization. Indeed, the GABA transporters have been implicated in epilepsy, and one isoform (GAT1) is the target of the anti-epileptic drug tiagabine. Unfortunately, no drug exists which specifically targets GAT2 or GAT3, but we have recently identified a GAT4-specific inhibitor. We will express the GABA transporters in Xenopus laevis oocytes in order to address the following Specific Aims: (1) To use rapid concentration jumps at the GABA transporter binding pocket in order to gain mechanistic insight about ion and substrate binding and translocation across the plasma membrane. These experiments will use a novel rapid perfusion system developed in this laboratory, and will perform Na +, CI-, and GABA jumps at the transporter to gain a detailed understating of ligand interaction with the transporter. (2) To fully examine a novel Cl- channel mode identified in GAT4. These experiments will illuminate the significance of a novel Cl- channel mode, which we have recently identified in GAT4. (3) To fully characterize the action of a recently identified inhibitor with selectivity for GAT4. We have succeeded in identifying a novel specific inhibitor for GAT4. The experiments of this aim will fully characterize the inhibitory action of this agent, and will pave the way for determining the contribution of GAT4 to GABAergic inhibitory neurotransmission. (4) To formulate a substrate pharmacophore for the GABA transporters GAT3 and GAT4. As most studies have focused on GAT1, very little is known about the substrate binding pocket of GAT3 and GAT4. The experiments of this aim will identify lead compounds for future structure-guided design of specific inhibitors of GAT3 and GAT4.